Linc-RAM is a metabolic regulator maintaining whole-body energy homeostasis in mice

Long noncoding RNAs (lncRNAs) are known to have profound functions in regulating cell fate specification, cell differentiation, organogenesis, and disease, but their physiological roles in controlling cellular metabolism and whole-body metabolic homeostasis are less well understood. We previously identified a skeletal muscle-specific long intergenic noncoding RNA (linc-RNA) activator of myogenesis, Linc-RAM, which enhances muscle cell differentiation during development and regeneration. Here, we report that Linc-RAM exerts a physiological function in regulating skeletal muscle metabolism and the basal metabolic rate to maintain whole-body metabolic homeostasis. We first demonstrate that Linc-RAM is preferentially expressed in type-II enriched glycolytic myofibers, in which its level is more than 60-fold higher compared to that in differentiated myotubes. Consistently, genetic deletion of the Linc-RAM gene in mice increases the expression levels of genes encoding oxidative fiber versions of myosin heavy chains and decreases those of genes encoding rate-limiting enzymes for glycolytic metabolism. Physiologically, Linc-RAM-knockout mice exhibit a higher basal metabolic rate, elevated insulin sensitivity and reduced fat deposition compared to their wild-type littermates. Together, our findings indicate that Linc-RAM is a metabolic regulator of skeletal muscle metabolism and may represent a potential pharmaceutical target for preventing and/or treating metabolic diseases, including obesity.


Introduction
Eukaryotic genomes are extensively transcribed to produce long noncoding RNAs (lncRNAs) in a temporally and spatially regulated manner [1,2]. An increasing number of lncRNAs have been reported to have profound functions in regulating cell lineage differentiation, cell proliferation, and tumorigenesis during development and in various pathological settings [3][4][5]. For example, in pluripotent cells, the divergent lncRNA Evx1as promotes the transcription of its neighboring gene, EVX1, to regulate mesendodermal differentiation [6]. The human-specific lincRNAs govern neuronal lineage commitment and contribute to human striatum development [7]. The lncRNA Handsdown (Hdn) regulates the cardiac gene program and is essential for early mouse development [8]. The myeloid-specific lncRNA LOUP originates from the upstream regulatory element of the PU.1 gene and induces myeloid differentiation by acting as a transcriptional inducer of the myeloid master regulator PU.1 [9].
Mechanistically, lncRNAs function as fundamental transcription and posttranscription regulators; and they act at multiple levels of gene expression in cis and/or trans in the nuclear and/or cytoplasmic compartments. Some experimental data suggest that lncRNAs function via regulating cell metabolism [10]. The lncRNA breast cancer anti-estrogen resistance 4 (BCAR4) reprograms glucose metabolism by upregulating the transcription of glycolysis-related genes in cancer cells [11]. The lncRNA GLCC1 is upregulated under glucose starvation in colorectal cancer cells to support cell survival and proliferation by enhancing glycolysis [12].
The lncRNA NEAT1 critically contributes to metabolic changes during breast cancer growth and metastasis by regulating the penultimate step of glycolysis [13]. LncRNA-ACOD1, which is identified by the nearby gene encoding aconitate decarboxylase 1 (Acod1), significantly attenuates viral infection by directly binding to the metabolic enzyme glutamic-oxaloacetic transaminase (GOT2) and enhancing its catalytic activity [14].
Skeletal muscle accounts for 40%-45% of the body mass and functions as an important metabolic and endocrine organ to orchestrate the basal metabolic rate [15,16]. It plays pivotal roles in regulating whole-body metabolic homeostasis by actively communicating with other metabolic organs, such as fat and liver [16][17][18]. Emerging studies have documented that lncRNAs regulate skeletal muscle cell differentiation during development and regeneration [19]. For example, the lncRNA Linc-MD1 has been shown to control muscle cell differentiation in both mouse and human myoblasts [20,21]. Although great progress has been made in elucidating the functions of lncRNAs in regulating muscle cell differentiation, we know relatively little about whether and how lncRNAs control muscle metabolism. Recent studies have shown that the lncRNA H19 acts to enhance muscle insulin sensitivity by activating AMPK [22]. The administration of H19 RNA gain-offunction oligonucleotides (H19-Rgof) was found to improve muscle mass, muscle performance, and the basal metabolic rate in mice. Furthermore, mice treated with H19 RNA reportedly resisted HFDor leptin deficiency-induced obesity [23].
We previously identified and characterized a long intergenic noncoding RNA (linc-RNA) activator of myogenesis (Linc-RAM), which is specifically expressed in skeletal muscle cells and localized in both the nucleus and the cytoplasm [24,25]. Nuclear Linc-RAM promotes muscle cell differentiation by facilitating the assembly of the MyoD-Baf60c-Brg1 complex on the regulatory elements of target genes [24]. Cytoplasmic Linc-RAM contributes to muscle cell differentiation by directly interacting with glycogen phosphorylase (PYGM) and modifying PYGM activity during myogenic differentiation [25]. Linc-RAM is transcriptionally regulated by MyoD via the FGF2/Ras/Raf/MEK/Erk signaling pathway during muscle cell differentiation [26]. However, the expression and physiological function(s) of Linc-RAM in fully differentiated mature skeletal muscle remain to be revealed.
In the present study, we report a physiological function of Linc-RAM in regulating skeletal muscle metabolism and the basal metabolic rate to maintain whole-body metabolic homeostasis. Our findings suggest that Linc-RAM may represent a potential pharmaceutical target for preventing and/or treating metabolic diseases, including obesity.

Mouse lines and animal care
All animal experiment procedures were approved by the Animal Ethics Committee of Peking Union Medical College (Beijing, China). Mice were housed in a pathogen-free facility and had free access to water and standard rodent chow under the following conditions: 21°C ambient temperature, 50%-60% humidity, and 12/12-h dark/ light cycle. The Linc-RAM-knockout mice in the C57BL/6j background were produced as previously described [24]. Two-monthold and 18-month-old male Linc-RAM-knockout and wild-type littermate mice were used in the study.

Primary myoblast isolation, culture, and differentiation
Primary myoblasts were isolated from hind-limb skeletal muscles of C57BL/6j mice at 2-3 weeks old, minced, and digested in a mixture of type II collagenase and dispase. Cells were filtered from debris and centrifuged, and fibroblasts were eliminated by differential attachment for 2×10 min. The obtained cells were cultured in F-10 Ham's medium (

Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR)
Trizol reagent (Invitrogen, Carlsbad, USA) was used to extract total RNA from proliferating myoblasts, differentiated myotubes, or various skeletal muscles, including gastrocnemius (Gas), quadriceps femoris (Qu), extensor digitorum longus (EDL), tibialis anterior (TA), and soleus (Sol). Total RNA was reverse-transcribed with reverse transcriptase (TaKaRa, Dalian, China). Real-time quantitative PCR analyses were performed in triplicate using Fast Eva Green qPCR Master Mix (Bio-Rad, Hercules, USA). β-Actin was used as an internal control for RT-qPCR analyses. All primers used for RT-qPCR are presented in Table 1.

Immunofluorescence staining
For cryosections of soleus muscle, the slides were incubated in 1.0% Triton X-100 in PBS at room temperature for 10 min. Subsequently, the sections were incubated at room temperature for 1 h in filtered blocking buffer (4% BSA, and 0.1% Triton X-100). The primary antibodies were diluted with PBS buffer containing 4% BSA. Monoclonal anti-myosin (skeletal, Fast) was purchased from Sigma (M1570; 1:200; St Louis, USA). Primary antibodies were loaded onto a specimen and incubated overnight at 4°C. Then, the slides were washed with PBS containing 0.1% BSA and incubated for 1 h with fluorescein-conjugated secondary antibodies (1:200; Zhongshanjinqiao Corporation, Beijing, China). After wash several times with PBS, the samples were imaged under a fluorescence microscope (Olympus, Tokyo, Japan).

Metabolic chamber analysis
Metabolic phenotyping of standard diet-fed wild-type and Linc-RAM-knockout mice was performed with the Oxymax/CLAMS metabolic cage system (Columbus Instruments, Columbus, USA) at the Animal Center of Peking Union Medical College. The mice had free access to water and standard rodent chow under a 12/12 h dark/light cycle. Food intake, drinking, O 2 consumption, and CO 2 production were automatically collected for 4 consecutive days.

Glucose-and insulin-tolerance tests
Overnight-fasted mice were given intraperitoneal (i.p.) injections of glucose (2 mg/g body weight) for the glucose tolerance test (GTT).
For the insulin tolerance test (ITT), mice were fasted for 4 h and then given 1 mU insulin/g body weight (Novolin, Tianjin, China) by i.p. injection. Blood glucose was determined with a Lifescan One Touch glucometer (Cat. No. G7021-1KG; Sigma).

Statistical analysis
Data are presented as the mean±SEM. For statistical comparisons of two conditions, the two-tailed Student's t test was used. Statistical analyses were performed using GraphPad Prism software. P<0.05 was considered statistically significant.

Linc-RAM is predominantly expressed in type II-enriched muscle groups of mice
We previously identified a skeletal muscle-specific lncRNA, Linc-RAM, which functions in regulating muscle cell differentiation [24].
To investigate the physiological function of Linc-RAM in mature skeletal muscle, we examined Linc-RAM expression in undifferentiated myoblasts, differentiated myotubes, and fully differentiated mature skeletal muscle (tibialis anterior). First, MyoG and myosin heavy chain (MyHC) expressions indicated that the cells were well differentiated ( Figure 1A,B). Linc-RAM was expressed in proliferating myoblasts cultured in growth medium (GM) and significantly upregulated when the cells were shifted to differentiation medium (DM) to undergo differentiation ( Figure 1C). These findings were consistent with our previous observations [24]. We further found that the expression level of Linc-RAM was 60-fold higher in mature skeletal muscle than in 2-day differentiated myotubes ( Figure 1C), suggesting that Linc-RAM is not functionally restricted to the regulation of muscle cell differentiation but rather may also play a role in mature skeletal muscle. Skeletal muscle comprises two types of myofibers that are distinguished by their contraction features: type I (slow-twitch) and type II myofibers (fast-twitch) [27]. The percentages of the myofiber types differ across various muscle groups and can adaptively change under physiological or pathological conditions [27]. To understand the physiological function of Linc-RAM in mature skeletal muscle, we examined its expression in various muscle groups from mice, including gastrocnemius (Gas), quadriceps femoris (Qu), extensor digitorum longus (EDL), tibialis anterior (TA), and soleus (Sol). We found that Linc-RAM was highly expressed in Gas, Qu, EDL, and TA muscles ( Figure 1D), which are enriched for type II myofibers [28]. In contrast, its expression level was low in Sol muscle, which is enriched for type I myofibers ( Figure 1D,E). Together, these findings indicate that Linc-RAM is predominantly expressed in type II-enriched muscle groups of mice.

Linc-RAM regulates fiber type and muscle metabolism in mice
The preferential expression of Linc-RAM in type II-enriched muscle groups suggested that Linc-RAM may function in regulating the muscle fiber type. We therefore analyzed fiber-type changes in the soleus muscle from Linc-RAM-knockout (KO) mice and compared to those of wild-type (WT) littermates at 2 months of age (young adult stage) (  Figure  3A-D). This finding indicated that Linc-RAM knockout decreased type IIx and type IIb myofibres at both the young and aged stages, and increased type IIa myofibres at the young stage but increased type I myofibres at the aged stage. To further corroborate the fiber type alteration in Linc-RAM-KO mice, we performed immunofluorescence staining of fast-twitch myofibers on cryosections of soleus muscle from the KO and WT mice at the aged stage ( Figure 3E). We found that the percentage of type I myofibers was significantly increased in the KO mice compared to the WT controls ( Figure 3F), whereas the percentage of type II myofibers was significantly decreased in the KO mice compared to that in the WT controls ( Figure 3G).
Given that type IIx and type IIb fibers are associated with active glycolytic metabolism [27], we next measured whether Linc-RAM knockout decreases glycolytic activity in skeletal muscle. To this end, we assayed the expression levels of genes encoding three ratelimiting enzymes of the glycolytic pathway: hexokinase 2 (HK2), muscle type phosphofructokinase (PFKm), and muscle type pyruvate kinase (PKm). The expression levels of HK2 and PFKm were significantly decreased in Linc-RAM-KO mice compared to Collectively, our data reveal that Linc-RAM regulates fiber type and muscle metabolism in mice.

Linc-RAM knockout slightly increases O 2 consumption and elevates insulin sensitivity at the young adult stage
To test whether Linc-RAM-mediated muscle metabolism plays a role in maintaining whole-body metabolic homeostasis, we next performed metabolic chamber analysis on Linc-RAM-KO mice and WT littermates at the young adult stage. The two groups of mice did not differ overtly in body weight ( Figure 4A), muscle mass ( Figure  4B), or fat mass ( Figure 4C). No significant differences were found in the amounts of food intake ( Figure 4D), water intake ( Figure 4E) and activity level ( Figure 4F) between the two groups of mice.
Interestingly, metabolic chamber analysis demonstrated that Linc-RAM-KO mice had slight increases in O 2 consumption ( Figure 4G), CO 2 production ( Figure 4H), and energy expenditure (EE) ( Figure  4I). Given that skeletal muscle functions as an important target organ of insulin signaling and plays pivotal roles in maintaining the blood glucose level [16], we performed glucose tolerance test (GTT) and insulin tolerance test (ITT) in the Linc-RAM-KO mice and WT littermates. We found that Linc-RAM-KO mice showed slightly improved glucose tolerance ( Figure 4J) and insulin sensitivity ( Figure 4K) compared to WT littermates. Thus, our experimental data reveal that Linc-RAM regulates glucose metabolism in skeletal muscle in mice.

Deletion of Linc-RAM increases the basal metabolic rate and reduces fat deposition in aged mice
Next, we examined whether Linc-RAM knockout influences wholebody metabolic homeostasis in 18-month-old Linc-RAM-KO mice and WT littermates fed with standard diet. We found that Linc-RAM KO mice exhibited a significantly reduced fat mass compared to WT littermates ( Figure 5A) and that this lean phenotype was not due to any between-group difference in the amount of food intake, water intake ( Figure 5B,C) or activity level ( Figure 5D). To further explore the mechanism underlying the lean phenotype of the Linc-RAM-KO mice, we conducted metabolic chamber analysis on the aged mice. Our results revealed that Linc-RAM-KO Linc-RAM is a metabolic regulator 1687 mice exhibited significantly increased O 2 consumption ( Figure 5E, H), CO 2 production ( Figure 5F,I), and energy production (EE) ( Figure 5G,J) compared to their WT littermates. As the lean phenotype is generally positively correlated with insulin sensitivity [16], we performed GTT and ITT in the aged Linc-RAM-KO mice and WT littermates. The GTT results showed that Linc-RAM-KO mice had significantly improved glucose tolerance ( Figure 5K) and significantly increased insulin sensitivity ( Figure  5L) compared to WT littermates. Together, our findings indicate that deletion of Linc-RAM enhances fat combustion and contributes to the lean phenotype observed in the aged Linc-RAM-KO mice, suggesting that Linc-RAM plays a regulatory role in maintaining whole-body metabolic homeostasis by controlling the basal metabolic rate in aged mice.

Discussion
Recent studies have documented that lncRNAs act as key regulators of cell differentiation, cell lineage choice, organogenesis, and tissue homeostasis [3,4]. However, the physiological roles of lncRNAs in regulating cellular metabolism and whole-body metabolic homeostasis are less well understood. Here, we report that Linc-RAM has a physiological function in regulating skeletal muscle metabolism and the basal metabolic rate to maintain whole-body metabolic homeostasis.
First, we demonstrated that Linc-RAM is preferentially expressed in type II-enriched muscle groups under physiological conditions. We previously observed that Linc-RAM is regulated by the transcription factor MyoD [24], which is expressed at a higher level in type II-enriched glycolytic TA muscle than in type I-enriched oxidative Sol muscle [29]. Accordingly, the expression level of Linc-RAM is significantly reduced in MyoD-knockout muscle [24,26]. Consistent with these expression patterns, we herein found that, similar to MyoD, Linc-RAM functions to regulate muscle fiber type. We show that Linc-RAM-knockout mice exhibit elevated expressions of genes encoding the oxidative myofiber versions of myosin heavy chain, Myh2 in young mice or Myh7 in aged mice, suggesting that deletion of Linc-RAM consistently increases oxidative metabolism and decreases glycolytic metabolism in both young and aged mice. Previous work revealed that MyoD gene knockout also increases the percentage of oxidative myofibers [30]. Yu et al. [31] demonstrated that lncRNA-FKBP1C regulates muscle fiber type by directly interacting with MYH1B and enhancing its protein stability. In contrast to the functions of Linc-RAM and MyoD, knockdown of LncRNA-FKBP1C was found to drive a fiber type switch from slowtwitch muscle fibers to fast-twitch muscle fibers [31]. Thus, our data and previous reports collectively suggest that lncRNAs play

1688
Linc-RAM is a metabolic regulator important roles in regulating myofiber type and skeletal muscle metabolism. Skeletal muscle, as an important metabolic and endocrine organ, plays pivotal roles in regulating whole-body metabolic homeostasis by actively communicating with other metabolic organs via muscleliver or muscle-fat crosstalk [16][17][18]. Here, we found that Linc-RAM-mediated muscle metabolism controls the basal metabolic rate and maintains metabolic homeostasis. We also revealed that Linc-RAM-knockout mice exhibit a higher basal metabolic rate, elevated insulin sensitivity, and reduced fat deposition than their wild-type littermates, highlighting that Linc-RAM-mediated muscle metabolism plays critical roles in orchestrating whole-body metabolic homeostasis. Previous work showed that the skeletal muscleenriched lncRNA H19 enhances muscle insulin sensitivity by

1690
Linc-RAM is a metabolic regulator Linc-RAM is a metabolic regulator 1691 activating AMPK [22] , and the administration of H19 RNA increases the basal metabolic rate and protects against high-fat diet (HFD)-or leptin deficiency-induced obesity [23]. A recent study showed that the mouse lncRNA Pair and human HULC, which are associated with phenylalanine hydroxylase (PAH), are involved in the development of the inherited metabolic disorder phenylketonuria (PKU) [32]. Pair-knockout mice faithfully model human PKU, and targeting HULC significantly reduces PAH enzymatic activity in human induced pluripotent stem cell-differentiated hepatocytes [32]. Collectively, these findings suggest that lncRNAs may represent potential pharmaceutical targets for preventing and/or treating metabolic diseases, such as obesity, as well as inherited metabolic disorders. Finally, our findings suggest an intriguing avenue through which researchers may identify signals or molecules that mediate the muscle-fat crosstalk for the lean phenotype observed in Linc-RAMknockout mice. Previous studies suggested that endogenous metabolites regulate whole-body metabolic homeostasis by mediating interorgan crosstalk. For example, one study showed that an insufficient alanine supply mediates muscle-liver-fat signaling by upregulating FGF21 expression in the liver [33]. Further identification of such mediator(s) would greatly improve our understanding of the molecular mechanism underlying interorgan crosstalk for the maintenance of whole-body metabolic homeostasis, providing potential targets for the development of therapeutic drugs that can be used to prevent and/or treat metabolic diseases.